Construction and identification of eukaryotic expression vector of human full-length PLCgamma1 gene.

OBJECTIVE

To construct the eukaryotic expression vector of human full-length PLCgamma1 gene for further study of the role of PLCgamma1 in cancer invasion.

METHODS

Reverse transcription-polymerase chain reaction (RT-PCR) technique was used to amplify human full-length PLCgamma1 gene from MG63 cells with a pair of specific primers containing the restriction sites for HindIII and NotI. After purification, the product of RT-PCR was digested with HindIII and NotI before insertion into the corresponding sites of eukaryotic expression vector pLNCX2, yielding the recombinant plasmid pLNCX2/PLCgamma1. PCR, restriction endonuclease analysis and DNA sequencing were performed to identify the recombinant eukaryotic expression vector pLNCX2/PLCgamma1. RT-PCR and Western blotting were used to detect the expression of the PLCgamma1 gene in LoVo cells after transient transfection via Lipofectamine TM 2000.

RESULTS

A 3 878-bp full-length PLCgamma1 gene fragment was successfully amplified by RT-PCR and inserted into eukaryotic expression vector pLNCX2. After digestion by HindIII and NotI, the recombinant eukaryotic expression vector pLNCX2/PLCgamma1 yielded a 3 878-bp fragment (PLCgamma1 gene) and a 6 100 bp fragment (vector). HindIII-BglII digestion was also done to verify the correctness of the recombinant plasmid, resulting in the identification of the fragments as expected. Sequencing analysis further confirmed the results. In addition, RT-PCR and Western blotting verified that the PLCgamma1 could overexpress in LoVo cells after transfection with recombinant eukaryotic expression vector pLNCX2/PLCgamma1.

CONCLUSION

The recombinant eukaryotic expression vector pLNCX2/PLCgamma1 has been constructed successfully.